1. Field of the Invention
This invention relates to a polypeptide having phosphoenolpyruvate carboxylase activity which does not require acetyl coenzyme A for activation and is desensitized to feedback inhibition by aspartic acid, and to genes coding for this polypeptide. The invention also relates to recombinant DNA molecules containing these genes, to bacteria transformed with these genes, and to methods of producing amino acids using the transformed bacteria.
2. Related Art
Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) is an enzyme which is found in almost all bacteria and all plants. PEP carboxylase catalyzes the condensation reaction between the three carbon glycolytic intermediate PEP and carbon dioxide resulting in the formation of the four carbon oxaloacetate (OAA), a metabolic intermediate common to the tricarboxylic acid (TCA) cycle and to L-aspartic acid biosynthesis. The TCA cycle requires continuous replenishment of C4 molecules in order to replace the intermediates withdrawn for amino acid biosynthesis, and by playing an anaplerotic role in supplying OAA to the TCA cycle, the biotin-independent PEP carboxylase aids in fulfilling this function.
OAA is a very important substrate for the production of cell metabolites such as amino acids, especially the glutamate family, i.e., glutamate, arginine and proline, and the aspartate family, i.e., aspartate, lysine, methionine, threonine and isoleucine. By catalyzing the reaction which results in the formation of OAA, PEP carboxylase plays an important role in supplying organic acids by metabolic processes. For example, fermentive production of succinic acid from glucose by Escherichia coli was significantly increased by the over-expression of PEP carboxylase. See Millard, C., et al., Appl. Environ. Microbiol. 62:1808-1810 (1996). Accordingly, PEP carboxylase also plays an important role in the production of amino acids which are formed from glutamate and aspartate.
The amino acid is a compound which universally exists in cells as components of proteins. However, for the sake of economic energy metabolism and substance metabolism, its production is strictly controlled. This control is principally feedback control, in which the final product of a metabolic pathway inhibits the activity of an enzyme which catalyzes an earlier step of the pathway. PEP carboxylase also undergoes various regulations in expression of its activity.
For example, in the case of PEP carboxylase of microorganisms belonging to the genus Brevibacterium, Corynebacterium or the genus Escherichia, PEP carboxylase activity is inhibited by aspartic acid. See e.g., Mori, M., et al., J. Biochem. 98:1621-1630 (1985); O""Regan, M., et al., Gene 77:237-251 (1989). Therefore, the aforementioned amino acid biosynthesis, in which PEP carboxylase participates, is also inhibited by aspartic acid. However, PEP carboxylase activities from Corynebacterium microorganisms having decreased sensitivity to aspartic acid have been described. See Eikmanns, B. J., et al., Mol. Gen. Genet. 218:330-339 (1989).
In addition to being allosterically inhibited by aspartic acid, acetyl co-enzyme A (acetyl-CoA) is an allosteric activator of PEP carboxylase from Brevibacterium flavum and Escherichia coli, for example. See Mori, M., et al., J. Biochem. 98:1621-1630 (1985); Morikawa, M., et al., J. Biochem. 81:1473-1485 (1977). PEP carboxylases from other organisms that are not regulated by aspartic acid or acetyl-CoA have been reported. See Valle, F., et al., J. Indus. Microbiol. 17:458-462 (1996); O""Regan, M., et al., Gene 77:237-251 (1989); Vance, C., et al., Plant Physiol. 75:261-264 (1984).
Since the anaplerotic enzyme PEP carboxylase is critical to the maintenance of an optimal pool of OAA, and consequently determines the biosynthetic levels of amino acids deriving from OAA, one way of improving amino acid production by fermentation would be to manipulate the corresponding ppc gene. For example, the amplification of the ppc gene from Brevibacterium lactofermentum has been shown to improve the production of proline and threonine. See Sano, K., et al., Agric. Biol. Chem. 51:597-599 (1987).
Various techniques have been developed for efficient production in amino acid fermentation by using mutant strains converted to be insensitive to feedback control. However, there has been no report of utilizing a PEP carboxylase derived from a plant for fermentative production of amino acids of the aspartic acid or glutamic acid families or of utilizing a ppc gene derived from a coryneform bacterium which is integrated into microbial chromosomal DNA for fermentative production of amino acids of the same families in which the PEP carboxylase is not substantially regulated by acetyl-CoA or aspartic acid.
U.S. Pat. No. 4,757,009 (Sano et al.; Ajinomoto Company) discloses a process for producing an amino acid by fermentation which comprises cultivating in a culture medium a Corynebacterium or Brevibacterium strain carrying a recombinant DNA molecule comprising a plasmid having operationally inserted therein a gene coding for PEP carboxylase, wherein the gene is a chromosomal gene isolated from a Corynebacterium or a Brevibacterium strain carrying a PEP carboxylase gene and has a chromosomal gene coding for an amino acid, and isolating the amino acid from the culture medium. The Corynebacterium or Brevibacterium strain from which the gene coding for PEP carboxylase is isolated is a strain which exhibits weakened feedback inhibition by aspartic acid.
European Patent No. 358,940 (Bachmann et al.; Degussa Aktiengesellschaft) discloses a plasmid pDM6 that is introduced into Corynebacterium glutamicum DM58-1, which is deposited at the Deutsche Sammlung von Mikroorganismen (DSM) under DSM 4697, wherein the plasmid contains a genetic sequence comprising information coding for the production of a protein having PEP carboxylase activity. The ppc gene is isolated from a genomic bank of Corynebacterium glutamicum ATCC 13032, and the PEP carboxylase is not stimulated by acetyl-CoA. Also disclosed is a method of producing L-lysine, L-threonine, and L-isoleucine by fermentation which comprises culturing in an appropriate medium a host bacterium belonging to the genus Corynebacterium or Brevibacterium which contains plasmid pDM6, and recovering the L-amino acid from the medium.
U.S. Pat. No. 5,876,983 (Sugimoto et al.; Ajinomoto Company) discloses a method of producing an amino acid which comprises selecting a microorganism of the genus Escherichia containing a DNA sequence encoding a mutant PEP carboxylase desensitized to feedback inhibition by aspartic acid by growing Escherichia microorganisms in the presence of a wild-type PEP carboxylase inhibitor selected from the group consisting of 3-bromopyruvate, aspartic acid-xcex2-hydrazide and DL-threo-xcex2-hydroxyaspartic acid; culturing a microorganism of the genus Escherichia or coryneform bacteria transformed with the DNA sequence encoding a mutant PEP carboxylase in a suitable medium; and separating from the medium an amino acid selected from the group consisting of L-lysine, L-threonine, L-methionine, L-isoleucine, L-glutamic acid, L-arginine and L-proline.
Although there are many examples of culturing amino acid-producing bacteria by recombinant DNA techniques, high levels of amino acid productivity are not always achieved. Therefore, a need still continues to exist for a method of producing amino acids by fermentation in high titre and yields. A PEP carboxylase that is not substantially regulated by acetyl-CoA or aspartic acid could improve carbon flow from the three carbon intermediate PEP to the four carbon intermediate OAA. The improved flow could contribute to compounds derived from OAA and increase amino acid biosynthesis.
Accordingly, the present invention relates to a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the gene is capable of being expressed in a host microorganism, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
The present invention also relates to a recombinant DNA molecule comprising a plasmid and a gene encoding a polypeptide having PEP carboxylase activity operationally inserted therein, wherein the recombinant DNA molecule is capable of propagating and the gene is capable of being expressed in a host microorganism comprising the genus Escherichia, Corynebacterium and Brevibacterium, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
The present invention further relates to a host microorganism belonging to the genus Escherichia, Corynebacterium or Brevibacterium transformed with a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the gene is derived from a plant belonging to the class Monocotyledonae or Dicotyledonae or from a microorganism belonging to the genus Corynebacterium or Brevibacterium, wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid, and wherein the host microorganism transformed with the DNA fragment expresses the gene.
In another aspect of the present invention there is provided a method of producing an amino acid by fermentation. The method comprises cultivating a host microorganism belonging to the genus Escherichia, Corynebacterium or Brevibacterium in a suitable medium and isolating from the culture medium an amino acid, wherein the host microorganism is transformed with a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity, wherein the host microorganism expresses the gene, and wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid.
In addition, the present invention relates to a method of selecting a DNA fragment comprising a gene encoding a polypeptide having PEP carboxylase activity wherein the polypeptide does not require acetyl-CoA for activation and is desensitized to feedback inhibition by aspartic acid, to a method of increasing the rate of conversion of PEP to OAA, to a method of recycling carbon in a fermentation process, to a method of assimilating carbon in a fermentation process which does not require biotin, to a method of increasing the production of organic acids in a fermentation process, and to a method of increasing the production of amino acids in a fermentation process.